Several recent publications indicate that glioblastoma multiforme (GBM) tumors contain a subpopulation of stem-like cells called "brain tumor stem cells" (BTSC) which are capable of self-renewal and of reforming a tumor with similar histological characteristics upon serial transplantation in mice. In contrast, the remaining, non-stem cells within the tumor are incapable of reforming the tumor. These findings suggest that BTSC may be the most important cells within a GBM in which to understand molecular abnormalities regulating cell growth, as well as the most important cells in which to target these abnormalities with novel therapies. We have recently shown that high expression levels of the signal transduction enzyme sphingosine kinase (SphK), which is overexpressed in several types of cancer, correlate with shorter survival of patients with GBM. We've also shown that the EGF receptor, which is commonly overexpressed in gliomas, signals through SphK. Our long term goal is to understand how SphK contributes to the malignancy of GBM and whether therapies targeting SphK could be used to effectively treat these malignancies. Our current hypothesis is that SphK enhances the malignant behavior of brain tumor stem cells and contributes to the transformation of neural stem cells. We will investigate this hypothesis through the following specific aims. 1) Determine the role of SphK in GBM tumor stem cell growth, self renewal, survival, invasiveness and tumor formation. We will culture BTSC from fresh GBM tissue and determine whether BTSC expressing different levels of SphK differ in their malignant behaviors. In addition, we will determine the effects of siRNA targeting SphK and dominant negative SphK on the above aspects of BTSC in vitro and on tumor formation by BTSC in nude mice. 2) Determine whether overexpression of SphK with or without EGF receptor or mutant EGF receptor overexpression in mouse neural stem cells leads to altered growth and invasion properties and/or oncogenic transformation. We will use an established mouse neural stem cell line and primary mouse neural stem cells. We will overexpress SphK and wild type or mutant EGF receptor in these cells and determine the effect on in vitro correlates of malignancy, such as cell growth rate, colony formation in soft agar, and in vitro invasiveness, as well as on tumor formation in nude mice. PUBLIC HEALTH RELEVANCE: This project will determine whether the protein sphingosine kinase makes a type of cell within brain tumors, called a brain tumor stem cell, behave more malignantly by growing faster or spreading through brain tissue more. It will also determine whether inhibiting sphingosine kinase decreases the growth and spread of these tumors and thus could be useful as an effective therapy for this disease.